The present invention relates to (microbial) epoxide hydrolases and biocatalytic reactions yielding optically pure epoxides and diols.
Optically active epoxides and 1,2-diols are important building blocks for the production of a range of optically active compounds that are needed for the production of pharmaceuticals and other fine chemicals. A number of strategies are available for the production of optically active epoxides (Schurig et al. 1992). An example that involves a catalytic asymmetric reaction is the Katsuki-Sharpless oxidation of unactivated double bonds, by means of an optically active titanium tartrate complex. A limitation is that the double bond must be in the allylic position relative to the hydroxyl group; this is necessary for coordination of the catalyst. Other procedures for the epoxidation of unfunctionalized prochiral alkenes have been developed using metalated salen- and porphyrin complexes as catalysts (Schurig et al. 1992). These methods have a low selectivity for terminal epoxides and limited applicability due to high catalyst cost and low turnover numbers. In general, the above methods cannot be applied economically on a large scale.
Biocatalytic reactions yielding optically pure epoxides have been described (Beetham et al. 1993; Faber et al, 1996). Lipases can be used for the resolution of racemic esters of epoxyalcohols to produce both enantiomers of the epoxyalcohol. Similarly, lipases may be used for the stereoselective resolution of esters formed from haloalcohols and a carboxylic acid. By transesterification, enantiomerically pure alcohols can be obtained, which may be converted to epoxides.
Biocatalytic production of epoxides is also possible by using mono-oxygenases. These enzymes require an unsaturated substrate and molecular oxygen. Reducing cosubstrates are required, and in general whole cells must be used to make regeneration of the cofactor economically feasible. Haloperoxidase based routes are also known. Haloperoxidases may produce optically active haloalcohols from alkenes.
Enzymatic kinetic resolution is a possible technique for the production of optically active epoxides. Enzymes that hydrolyse epoxides are called epoxide hydrolases. They convert epoxides to diols by cleaving the epoxide ring with water. Epoxide hydrolases have been detected in mammals, insects, yeasts, fungi, other eukaryotic organisms, and in prokaryotic organisms.
The epoxide hydrolases from higher eukaryotic organisms play a role in endogeneous metabolism and do not show strong substrate selectivity. They convert a variety of compounds formed from drugs by oxidative enzymes such as cytochrome P450. The sequence similarity of some epoxide hydrolases to dehalogenases (Beetham et al. 1995, Arand et al. 1994) suggests that they may act by covalent catalysis via a mechanism that does not allow racemisation (Lacourciere et al.,1993; Pries et al., 1994). The epoxide ring is opened by a nucleophilic attack of an carboxylate residue, yielding a covalent ester intermediate. Subsequently this intermediate is hydrolysed and the diol is released.
Production of epoxide hydrolases by eukaryotic organisms is well known. Most work has been done with crude microsomal preparations of liver tissue from rats or other mammals (Wistuba et al. 1992). Mammalian epoxide hydrolases have been studied in detail because epoxides are important in toxicology and in chemical carcinogenesis (Guengerich, 1982). However, the enzymes from mammals, which are known to be stereoselective in some cases, are difficult to obtain and serve multiple functions in vivo.
Styrene oxides are well investigated substrates for microsomal epoxide hydrolases. They have been used in a standard assay for determinaton of the activity of the enzyme. Styrene oxides with substituents on the ring were investigated for studies on the mechanism of this enzyme (Dansette et al. 1978). Further research showed that the microsomal epoxide hydrolase was able to hydrolyse enantioselectivily styrene oxide (Watabe et al. 1983). The time course of this enzymatic hydrolysis showed a biphasic shape. At first the (R)-enantiomer is hydrolysed. After 90% conversion of the (R)-enantiomer, the (S)-enantiomer is hydrolysed at a much faster rate. This behaviour was explained by the fact that the (R)-enantiomer with a smaller Km could inhibit the hydrolysis of the faster reacting (S)-enantiomer (higher Km, higher Vmax). The enantioselective hydrolysis with mEH was also investigated with p-nitro styrene oxide (Westkaemper et al. 1980) and xcex2-alkyl substituted styrene oxides (Belluci et al 1993 and 1996). Fungal or yeast enzymes are also known and used experimentally. These also are likely to serve detoxification functions or are involved in endogeneous metabolism and are also not very selective. Recently, epoxide hydrolases from Aspergillus niger have been used to resolve successfully styrene oxide (Chen et al. 1993) and para substituted styrene oxides such as p-nitro styrene oxide and p-chloro styrene oxide with high enantioselectivity towards the S-enantiomer (Nelliah et al. 1996, Pedragosa-Moreau et al 1996). Another fungus, Beauveria sulfurescens, showed high R-selectivity for styrene oxide.
However, production in prokaryotic expression systems of substantial amounts of the epoxide hydrolases that originate from eukaryotes has shown not to be practically feasible. Although many of the above enzymes can be studied experimentally, none of them is available for industrial biocatalytic application. Typical industrial processes which would benefit, however, from the availability of well defined epoxide hydrolases would be the preparation of enantiopure epoxides and 1,2-diols as for example is useful in the production of pheromones and vitamins. Bacterial epoxide hydrolases have been detected in epichlorohydrin-degraders (Nakamura et al. 1994, Jacobs et al. 1991), in organisms that degrade epoxysuccinic acid (Hand et al. 1969), and in organisms that convert nitrils (Hechtberger et al. 1993). The bacterial epoxide hydrolases probably serve a function in the metabolism of endogenous compounds or epoxides produced by monooxygenases, and their selectivitity is not considered high. In a recent review on microbial epoxide hydrolases (Faber et al, 1996) it was observed that epoxide hydrolases could amply been found in eukaryotic cells but were rare in prokayotic cells. However, in some isolates from bacterial genera such as Corynebacterium, Pseudomonas, Bacillus, Mycobacterium and Rhodococcus epoxide hydrolase activity has been observed. Faber et al also described that epoxide hydrolase activity could be induced in only a limited number of bacterial isolates, notably in Rhodococcus spp and in Mycobacterium, whereas isolates from Corynebacterium and Pseudomonas spp did not show any epoxide hydrolase activity and epoxide hydrolase could not be induced by growing these latter species on a selective medium. In addition, Faber et al remark that, although prokaryotic epoxide hydrolases have some advantages above eukaryotic epoxide hydrolases, the enantioselectivities of microbial hydrolases (expressed as enantiomeric ratio (ee)) are low and highly substrate dependent. Faber further remarks, that only a few microbial strains possessing suitable epoxide hydrolase activity for a given substrate are known and prediction of suitable microbial strains is not yet possible, until, for example, the three-dimensional X-ray structure of an epoxide hydrolase has been solved.
The present invention now surprisingly provides isolated micro-organisms that express epoxide hydrolases with a high enantioselectivity. Said micro-organisms represented and provided for by the invention can selectively degrade epichlorohydrin or related halopropanol compounds and their genome encodes a polypeptide having a highly enantioselective epoxide hydrolase activity.
The isolated micro-organism which is the representative micro-organism provided by the invention is Agrobacterium spp exhibiting high enantioselectivity. The micro-organisms provided by the invention essentially correspond to the micro-organism represented by Agrobacterium radiobacter deposited under deposit number CBS 750.97. The terms xe2x80x9cessentially correspondxe2x80x9d or xe2x80x9cessentially correspondingxe2x80x9d refer to variations that occur in nature and to artificial variations of representative micro-organisms which can selectively degrade epichlorohydrin or related halopropanol compounds and are having enantioselective epoxide hydrolase activity. In particular said variations relate to variations in the epoxide hydrolase and functional fragments thereof which can be isolated from said micro-organisms and to variations in the genome of said organisms encoding epoxide hydrolase and functional fragments thereof. In particular, the terms xe2x80x9cessentially correspondxe2x80x9d or xe2x80x9cessentially correspondingxe2x80x9d relate to those variations that still allow detection of epoxide hydrolase or functional fragments thereof in tests for epoxide hydrolase activity or detection of the genome (be it DNA or RNA) or fragments thereof encoding epoxide hydrolase or functional fragments thereof by hybridization techniques, (such as nucleic acid blotting or in situ hybridization) or amplification techniques (such as PCR).
The invention also provides a pure culture of the micro-organism, and a (lyophilized) preparation thereof. The invention also provides a crude or pure enzyme preparation derived from said micro-organism, preparing and purifying enzymes from micro-organisms comprise ordinary skills known in the art. In addition, the invention provides (partly) purified epoxide hydrolase. The enzyme provided by the invention has a molecular weight of 30-40 kD as determined by SDS-polyacrylamide electrophoresis and contains 280-350 amino acids. Epoxide hydrolase as provided by the invention is generally water soluble, generally stable in Tris buffer at neutral pH and is generally able to convert epoxides with a broad specificity. It catalyses the conversion by covalent catalysis, using a nucleophilic aspartate.
The invention now also provides the isolated gene (a recombinant DNA molecule) or parts thereof encoding at least a functional part of a polypeptide having epoxide hydrolase activity. Such genes or fragments thereof can for example be derived from micro-organisms essentially corresponding to those that can selectively degrade epichlorohydrin or related halopropanol compounds.